Difference between revisions of "Part:BBa K510022:Experience"
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| + | =Construction of a "portable" attTn7= | ||
| + | As a first step toward this goal, we undertook the cloning of the functional E. coli attTn7 into a plasmid vector, and the demonstration that this "portable" plasmid-borne attTn7 can be recognized as a Tn7 transposition target by our miniTn7BB minitransposons. The attTn7 was PCR-amplified from the chromosome of the E. coli K12 strain MC4100 with primers bearing prefix and suffix restriction sites, cleaved and ligated into pSB1C3. The construct was '''verified by sequencing'''. | ||
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| + | =Characterization of the "portable" attTn7= | ||
| + | For preliminary characterization of the "portable" attTn7, we attempted to demonstrate transposition into the plasmid-borne target. The miniTn7BB-Gm transposon was transferred into E. coli DH5α bearing pSB1C3-attTn7 by co-electrotransformation with the helper plasmid pTNS2. Transformants bearing transposon insertions were selected on LB plates supplemented with gentamycin and chloramphenicol. To identify clones bearing the transposon insertion at the plasmid-borne target, colony PCR was performed using prefix and Tn7R primers. Unfortunately, the results of this assay are too preliminary to be shown. | ||
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===Applications of BBa_K510022=== | ===Applications of BBa_K510022=== | ||
Revision as of 23:33, 21 September 2011
Contents
Construction of a "portable" attTn7
As a first step toward this goal, we undertook the cloning of the functional E. coli attTn7 into a plasmid vector, and the demonstration that this "portable" plasmid-borne attTn7 can be recognized as a Tn7 transposition target by our miniTn7BB minitransposons. The attTn7 was PCR-amplified from the chromosome of the E. coli K12 strain MC4100 with primers bearing prefix and suffix restriction sites, cleaved and ligated into pSB1C3. The construct was verified by sequencing.
Characterization of the "portable" attTn7
For preliminary characterization of the "portable" attTn7, we attempted to demonstrate transposition into the plasmid-borne target. The miniTn7BB-Gm transposon was transferred into E. coli DH5α bearing pSB1C3-attTn7 by co-electrotransformation with the helper plasmid pTNS2. Transformants bearing transposon insertions were selected on LB plates supplemented with gentamycin and chloramphenicol. To identify clones bearing the transposon insertion at the plasmid-borne target, colony PCR was performed using prefix and Tn7R primers. Unfortunately, the results of this assay are too preliminary to be shown.
Applications of BBa_K510022
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